Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis

Abstract

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type alpha-subunits and with C-terminal deletion mutants of the alpha-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

FIG. 1.

Nucleotide sequence of the promoter region of the comG operon from bp −204 to +12. The nucleotides are numbered corresponding to the in vivo transcriptional start site from the comG promoter. In the comG promoter, three potential ComK dimer binding sites (AT boxes) are present (underlined). The site indicated in boldface and underlined is located at a position comparable to the ComK-binding site in other ComK-regulated promoters. The position of the starting nucleotide (italic) of the common AT box (position −86) is indicated, as well as the starting position of the extra AT box (position −109). The proposed −10 and −35 promoter sequences are printed in boldface. The B. subtilis σ^A consensus promoter is depicted underneath the comG sequence for comparison (bases in capital letters are present in >70% of the promoters) (36).

FIG. 2.

Complexes formed by RNAP and the comG promoter with or without ComK present. Electrophoretic mobility shift assays were performed with a 200-bp ³²P-labeled comG promoter fragment. RNAP concentrations were increased in twofold increments from 0 to 44 nM, as indicated by the concentration bars. The positions of the different complexes are shown on the right. K, ComK; R, RNAP; Fp, free probe. (A) RNAP binding in the absence of ComK. (B) RNAP binding in the presence of 0.35 μM ComK. For comparison, a blank sample (−) and binding of only RNAP (R; 22 nM) are shown in the first and second lanes, respectively. (C) The percentage of radioactive probe in the RNAP-promoter complex as determined by densitometric scanning was plotted against nanomoloar RNAP concentration. The percentage was calculated by dividing the signal of the RNAP band by the total signal in each lane, which was determined by combining the intensities of the bands present per lane. Triangles, no ComK present; squares, 0.35 μM ComK.

FIG. 3.

The upstream region of the comG promoter is responsible for stabilizing the RNAP-promoter complex. (A) Electrophoretic mobility shift experiments were performed with a ³²P-labeled truncated comG (comG_trn, two AT boxes, no upstream DNA) promoter fragment. Reaction mixtures contained RNAP (17.5 nM) and/or ComK (0.35 μM), as indicated above the lanes. The positions of the different complexes are shown on the right. K, ComK; R, RNAP; Fp, free probe. (B) In vitro transcription reactions were performed as described in Materials and Methods on pAN-G or derivatives containing wild-type (wt) upstream DNA or nonspecific upstream DNA, as indicated in panel C. AT2, two AT boxes; AT3, three AT boxes; 1, no ComK present; 2, 0.07 μM ComK; 3, 0.35 μM ComK. (C) Schematic overview of the constructs tested for the influence of the upstream DNA on stabilization of RNAP binding (Stab. bind.) and transcription (Transcript). n.d., not determined. The AT boxes are represented as squares, and the RNAP-binding site is represented as a rectangle. The fragments contain upstream DNA (300 bp) of wild-type comG origin, high-GC DNA, high-AT DNA, or no upstream DNA. The dotted lines indicate at which position of the wild-type comG promoter the fusions or truncations were made. (The numbers correspond to those in Fig. 1.) The situations with nonspecific upstream DNA or no upstream DNA were tested for both three and two AT boxes. ++, wild-type stabilization of RNAP binding or transcription in the presence of ComK (100%); +, 40 to 80% stabilization; −, 0 to 10% stabilization.

FIG. 4.

ComK stimulation of transcription by RNAP containing wild-type (Wt) or mutant α-subunits. (A) In vitro transcription reactions were performed as described in Materials and Methods. Products were separated by electrophoresis. The constitutive C2 promoter from phage φ29 was used as an internal standard for specific activity of reconstituted RNAPs. (B) Transcription products were quantified with a Fuji BAS-IIIs image analyzer. Transcription is reported as fold stimulation over that with no ComK added for each RNAP preparation, corrected for differences in specific RNAP activity by use of the internal standard. The abbreviations RP-wt, RP-15, RP-37, and RP-59 represent RNAP reconstituted with the wild-type α-subunit and the Δ15, Δ37, and Δ59 deletion mutants, respectively.

FIG. 5.

(A) Requirement of initiating nucleotides for heparin resistance. Electrophoretic mobility shift experiments were performed with the ³²P-labeled comG promoter fragment. (B) Helix face dependency of the ComK effect on RNAP binding to the promoter. Electrophoretic mobility shift experiments were performed with the ³²P-labeled comG+6 (206-bp) promoter fragment. For both panels A and B, reaction mixtures contained RNAP (17.5 nM), ComK (0.35 μM), and initiating nucleotides (200 μM) as indicated above the lanes. Heparin challenge was performed as described in Materials and Methods. The positions of the different complexes are shown on the right. K, ComK; R, RNAP; H, heparin; A, ATP; G, GTP; fp, free probe; a, aspecific binding in lane 2, connected with the purified RNAP sample.

FIG. 6.

Model of transcription activation by ComK at the comG promoter. ComK proteins are represented as the small circles, RNAP is represented as a large ellipsoid, and DNA is represented by a solid line. For details, see the text.